Crystalline form of triethylenetetramine tetrahydrochloride and its pharmaceutical use

ABSTRACT

The present invention describes a new crystalline form of triethylenetetramine tetrachloride which has improved room temperature stability over known forms and over the dichloride salt. The new crystalline form is characterised by having peaks in an XRPD spectrum at 22.9, 25.4, 25.8, 26.6, 34.6 and 35.3±0.1°2θ and Raman shifts 943, 1173, 1527 and 1612±5 cm −1 . The crystalline form of triethylenetetramine tetrachloride is useful in the treatment of Wilson&#39;s disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/EP2019/061441, filed on May 3, 2019, which in turn claims thebenefit of Application No. EP 18290048.0, filed on May 4, 2018. Theseapplications are incorporated herein in their entireties.

FIELD OF THE INVENTION

The invention relates to a crystalline form of triethylenetetraminetetrachloride (TETA.4HCl) and methods of making the crystalline form.The invention further relates to treatment of Wilson's disease using thecrystalline triethylenetetramine tetrachloride.

BACKGROUND TO THE INVENTION

Triethylenetetramine, or 1,2-ethanediamine, N, N′-bis(2-aminoethyl)(TETA) has the structure:

The dichloride salt (TETA.2HCl) is a polyamine chelator of copper (II).Its copper chelating properties make it useful in the treatment ofvarious conditions, in particular Wilson's disease. Wilson's disease isa genetic disorder caused by a mutation in the Wilson disease protein(ATP7B gene). The condition leads to a build up of copper in the body.The copper chelating ability of TETA.2HCl also led to its considerationfor the treatment of numerous conditions such as internal organ damagein diabetes patients, Alzheimer's disease and cancer (Henriet et al,International Journal of Pharmaceutics 511 (2016) 312-321).

However, despite the many years over which TETA.2HCl has been known tobe useful for the treatment of Wilson's disease, it has not been asuccessful treatment. This is, at least in part, because it has provendifficult to provide suitable forms of TETA.2HCl which have sufficientstability at room temperature. It is therefore necessary for patients tostore tablets under reduced temperature conditions, an onerousrequirement for a treatment which needs to be taken with every meal, forlife.

Studies have also shown that variation in humidity can affect thestability of the salt. The salt is very sensitive to water and exists indifferent polymorphic forms dependent on the humidity levels. Highhumidity results in instability of the compound. These stability effectslead to challenges in the formulation of a suitable drug for thetreatment of patients and the need to store materials under specialconditions such as reduced temperature. There is therefore a need forimproved treatments for Wilson's disease which can be delivered orallyand which are stable under ambient conditions over long periods of time.

EP 1778618 describes synthetic techniques for producing TETA and itssalts including the 0.2HCl salt and the 0.4HCl salt. Only the 2HCl saltis said to be useful in the treatment of Wilson's disease.

WO 2006/027705 describes the synthesis of triethylenetetramines,including Form I and Form II triethylenetetramine dihydrochloride. Thisdocument does not mention the crystalline forms of triethylenetetraminetetrahydrochloride.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that a new crystallineform of TETA.4HCl has improved handling properties and room temperaturestability. It is therefore more useful for formulation into a drug thaneither the dichloride or known forms of the tetrachloride salt.Previously known techniques for producing TETA.4HCl (such asanti-solvent crystallisation processes carried out at room temperature,and processes including high temperature drying steps) lead to acrystalline form described herein as Form A. The present inventors,however, have found that by carefully controlling the conditions ofmanufacture, in particular the temperature and rate of crystallisation,a new crystalline form, known herein as Form B, can be produced. Thisnew form has good handling properties and also good stability and shelflife characteristics and is therefore beneficial in the production ofnew formulations, for example tablets, for treating Wilson's disease.

The present invention therefore provides a crystalline form oftriethylenetetramine tetrachloride having at least one of the followingcharacteristics:

(i) an XRPD pattern having at least two peaks selected from the peaks at22.9, 25.4, 25.8, 26.6, 34.6 and 35.3±0.1°2θ; and/or

(ii) a Raman spectrum having at least two peaks selected from the peaksat a Raman shift of 943, 1173, 1527 and 1612±5 cm⁻¹.

Also provided is a pharmaceutical composition comprising the crystallineform as described herein together with one or more pharmaceuticallyacceptable carriers or diluents.

Also provided is a method of producing a crystalline form oftriethylenetetramine tetrachloride which comprises adding ananti-solvent to an aqueous solution of triethylenetetraminetetrachloride and collecting the crystals obtained, wherein theanti-solvent addition is carried out at a temperature of about 20° C. orbelow.

Also provided is a crystalline form of triethylenetetraminetetrachloride, or a pharmaceutical composition containingtriethylenetetramine tetrachloride, obtainable or obtained by themethods described herein.

Also provided is a crystalline form or pharmaceutical composition asdescribed herein for use in the treatment of the human or animal body bytherapy, preferably for use in the prevention or treatment of Wilson'sdisease.

Also provided is a method for the prevention or treatment of Wilson'sdisease in a subject in need thereof, which method comprises theadministration to the subject of an effective amount of the crystallineform or pharmaceutical composition as described herein.

Also provided is the use of a crystalline form or pharmaceuticalcomposition as described herein in the manufacture of a medicament forthe prevention or treatment of Wilson's disease.

Particular aspects of the invention are set out below:

1. A crystalline form of triethylenetetramine tetrachloride having atleast one of the following characteristics:

(i) an XRPD pattern having at least two peaks selected from the peaks at22.9, 25.4, 25.8, 26.6, 34.6 and 35.3±0.1°2θ; and/or

(ii) a Raman spectrum having at least two peaks selected from the peaksat a Raman shift of 943, 1173, 1527 and 1612±5 cm⁻¹.

2. A crystalline form according to aspect 1, having an XRPD patternhaving at least two peaks selected from the peaks at 22.9, 25.4, 25.8,26.6, 34.6 and 35.3±0.1°2θ.

3. A crystalline form according to aspect 1 or aspect 2, having an XRPDpattern having at least three peaks selected from the peaks at 22.9,25.4, 25.8, 26.6, 34.6 and 35.3±0.1°2θ.

4. A crystalline form according to any one of aspects 1 to 3, having anXRPD pattern having peaks at 25.4, 34.6 and 35.3±0.1°2θ.

5. A crystalline form according to any one of aspects 1 to 4, containingno more than 10 wt % of triethylenetetramine tetrachloride Form A havingan XRPD pattern having peaks at 25.2 and 35.7±0.10°2θ.

6. A crystalline form according to aspect 5 which contains no more than5 wt % of triethylenetetramine tetrachloride Form A having an XRPDpattern having peaks at 25.2 and 35.7±0.1°2θ.

7. A crystalline form according to any one of the preceding aspectswhich consists essentially of triethylenetetramine tetrachloride Form Bhaving:

-   (i) an XRPD pattern as defined in any one of claims 1 to 4; and/or-   (ii) a Raman spectrum having at least two peaks selected from the    peaks at a Raman shift of 943, 1173, 1527 and 1612±5 cm⁻¹.    8. A pharmaceutical composition comprising a crystalline form    according to any one of aspects 1 to 7 and a pharmaceutically    acceptable carrier or diluent.    9. A pharmaceutical composition according to aspect 8, which is a    solid oral dosage form comprising a crystalline form according to    any one of claims 1 to 7 and a pharmaceutically acceptable carrier.    10. A pharmaceutical composition according to aspect 8 or aspect 9,    which contains no more than 10 wt %, preferably no more than 5 wt %,    more preferably no more than 2 wt % triethylenetetramine    tetrachloride Form A having an XRPD pattern having peaks at 25.2 and    35.7±0.1°2θ.    11. A pharmaceutical composition according to aspect 10 which is    substantially free of triethylenetetramine tetrachloride Form A    having an XRPD pattern having peaks at 25.2 and 35.7±0.1°2θ.    12. A method of producing a crystalline form of triethylenetetramine    tetrachloride, which method comprises adding an anti-solvent to an    aqueous solution of triethylenetetramine tetrachloride and    collecting the crystals obtained, wherein the anti-solvent addition    is carried out at a temperature of about 20° C. or below.    13. A method according to aspect 12, wherein the rate of addition of    anti-solvent to the solution is no more than 0.5 ml/min per gram of    TETA.4HCl dissolved in the aqueous solution.    14. A method according to aspect 12 or aspect 13, which method    comprises:-   (i) adding anti-solvent to an aqueous solution of TETA.4HCl at    temperature T1 over a period of time t1 and/or at a rate of addition    R1;-   (ii) optionally adding TETA.4HCl seed crystals;-   (iii) optionally agitating the resulting mixture at T1 for a further    period t1 a;-   (iv) optionally reducing the temperature to temperature T2 and    agitating the mixture for a further period t2; and-   (v) collecting the resulting crystals;    wherein T1 is about 20° C. or below; T2 is at least 5° C. lower than    T1; t1 is at least 1 hour, R1 is 0.5 ml/min/g or less, t1 a is at    least 2 hours and t2 is at least 30 minutes.    15. A method according to aspect 14, which method comprises:-   (i) adding anti-solvent to an aqueous solution of TETA.4HCl at    temperature T1 over a period of time t1 and or at a rate R1;-   (ii) optionally adding TETA.4HCl seed crystals;-   (iii) optionally agitating the resulting mixture at T1 for a further    period t1 a;-   (iv) reducing the temperature to temperature T2 and agitating the    mixture for a further period t2; and-   (v) collecting the resulting crystals;    wherein T1 is about 20° C. or below; T2 is about 10° C. or below; t1    is at least 1 hour, R1 is 0.5 ml/min/g or less, t1 a is at least 3    hours and t2 is at least 30 minutes.    16. A method according to aspect 15, which method comprises:-   (i) adding anti-solvent to an aqueous solution of TETA.4HCl at    temperature T1 over a period of time t1 and/or at a rate R1;-   (ii) adding TETA.4HCl seed crystals;-   (iii) agitating the resulting mixture at T1 for a further period    tla;-   (iv) reducing the temperature to temperature T2 and agitating the    mixture for a further period t2; and-   (v) collecting the resulting crystals;    wherein T1 is about 15° C. or below; T2 is about 5° C. or below; t1    is at least 1 hour, R1 is 0.2 ml/min/g or less, t1 a is at least 4    hours and t2 is at least 30 minutes.    17. A method according to any one of aspects 12 to 16 which further    comprises drying the collected crystals at a temperature of below    about 40° C., preferably below about 30° C.    18. A method according to any one of aspects 12 to 17 wherein the    collected crystals are combined with a pharmaceutically acceptable    carrier to produce a pharmaceutical composition.    19. A method according to aspect 18, wherein the method further    comprises compressing the mixture of crystals and pharmaceutically    acceptable carrier to form a tablet and optionally sugar-coating or    film-coating the tablet.    20. A crystalline form or pharmaceutical composition obtainable by    the method of any one of aspects 12 to 19.    21. A crystalline form or pharmaceutical composition according to    any one of the preceding aspects, for use in a method of treating    the human or animal body by therapy.    22. A crystalline form or pharmaceutical composition for use    according to aspect 21, which is for use in preventing or treating    Wilson's disease.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1a and 1b are flow charts showing a method for producing TETA.4HClin crude form.

FIG. 2 is a flow chart showing a method for producing TETA.4HCl Form Bin substantially pure form.

FIG. 3 depicts the X-ray powder diffraction pattern of TETA.4HCl Form A(“Profile 1”) and of a mixture of TETA.4HCl Forms A and B (“Profiles1+2”). Arrows highlight the characteristic peaks of TETA.4HCl Form B.

FIG. 4 depicts the X-ray diffraction pattern of TETA.4HCl Form B.

FIG. 5a shows the Raman spectra of TETA.4HCl Form A (below) and Form B(above).

FIG. 5b shows the spectra overlaid with Form A in the upper line andForm B the lower line.

Form B peaks are highlighted.

FIG. 6 depicts a Heckel plot of TETA.4HCl Form A and TETA.4HCl Form B.

FIG. 7a shows a tablet of TETA.4HCl formed from TETA.4HCl Form A. FIG.7b shows an image of the same tablet after aging. FIG. 7c shows a Ramananalysis of the aged tablet, with the darkened regions corresponding tothe location of TETA.4HCl Form A in the tablet.

FIGS. 8a and 8b show the FTIR-ATR spectra of TETA.4HCl Form B. FIG. 8ashows the full spectrum 4000-525 cm⁻¹. FIG. 8b shows the fingerprintregion 1800-525 cm⁻¹.

FIG. 9a shows X-ray Powder diffraction analyses on the samples ofExample 7 after manual addition of ethanol at different speeds, andcomparison with the reference patterns of Form A and Form B. FIG. 9bshows magnification of FIG. 9a on 2θ=20-29°. FIG. 9c shows magnificationof FIG. 9a on 2θ=34-37°.

FIG. 10a shows X-ray Powder diffraction analyses on the samples ofExample 7 after programmed addition of ethanol, and comparison with thereference patterns of Form A and Form B. FIG. 10b shows magnification ofFIG. 10a on 2θ=20-29°. FIG. 10c shows magnification of FIG. 10a on2θ=34-37°.

FIGS. 11a and 11b show the change in mass with respect to relativehumidity for DVS analysis of Example 2 and Reference Example 3respectively.

FIGS. 12a to 12c show XRPD spectral analysis of a product produced inaccordance with a prior art process.

DETAILED DESCRIPTION OF THE INVENTION

Crystalline TETA.4HCl

The crystalline form of triethylenetetramine tetrachloride (TETA.4HCl)which is described herein is known as Form B. This crystalline form canbe characterised by one or more of its XRPD spectrum, its Ramanspectrum, its melting point, its FTIR spectrum and its DVS behaviour.Details of each of these characteristics of the crystalline form aredescribed below. Typically, the crystalline form of the invention ischaracterised by its XRPD spectrum and/or its Raman spectrum, mostpreferably its XRPD spectrum. Thus, the crystalline form of theinvention typically has at least one of the following characteristics:

(i) an X-ray powder diffraction (XRPD) pattern having at least two peaksselected from the peaks at 22.9, 25.4, 25.8, 26.6, 34.6 and 35.3±0.1°2θ;and/or

(ii) a Raman spectrum having at least two peaks selected from the peaksat a Raman shift of 943, 1173, 1527 and 1612±5 cm⁻¹.

Typically, the crystalline form of TETA.4HCl of the invention has anXRPD pattern having at least two peaks selected from the peaks at 22.9,25.4, 25.8, 26.6, 34.6 and 35.3±0.1°2θ. Preferably, the XRPD pattern hasat least three peaks, more preferably at least four peaks selected fromthe peaks at 22.9, 25.4, 25.8, 26.6, 34.6 and 35.3±0.1°2θ. Morepreferably, at least 5 or all of these peaks are observed in the XRPDpattern. More preferably, the crystalline form of TETA.4HCl of theinvention has an XRPD pattern having at least two peaks, preferably atleast three, four, five or all of the peaks, selected from the peaks at22.9, 25.4, 25.8, 26.6, 34.6 and 35.3±0.05°2θ It is particularlypreferred that the crystalline form of TETA.4HCl has an XRPD patternhaving peaks at 25.4, 34.6 and 35.3±0.1°2θ, more preferably at 25.4,34.6 and 35.3±0.05°2θ.

Typically, the peaks at 25.4 and 35.3±0.1°2θ are the most intense, inparticular the peak at 25.4±0.1°2θ. Preferably, the peak at 25.4±0.1°2θis at least twice as intense as the next most intense peak, morepreferably at least three times as intense. Typically, the peak at35.3±0.1°2θ is at least twice as intense as the next most intense peak.

Typically, the XRPD pattern of TETA.4HCl Form B is substantially similarto that depicted in FIG. 4.

XRPD data can be obtained using the PANALYTICAL X'PERT PRO MPDdiffractometer. Diffraction data is typically acquired by exposingpowder samples to Cu—K_(α) X-ray radiation, which has a characteristicwavelength (λ) of 1.5418 Å. X-rays were generated from a Cu anodesupplied with 40 kV and a current of 40 mA. Further details of operatingconditions for obtaining XRPD data are set out in the Examples sectionherein.

Typically, the crystalline form of TETA.4HCl of the invention has aRaman spectrum having shifts at two or more of 943, 1173, 1527 and1612±5 cm⁻¹. Preferably, the Raman spectrum shows at least two,preferably three, more preferably all four of the peaks at 943, 1173,1527 and 1612±5 cm⁻¹. It is particularly preferred that the crystallineform of TETA.4HCl has a Raman spectrum having shifts at two or more,preferably three, more preferably all four, of 943, 1173, 1527 and1612±2 cm⁻¹. It is particularly preferred that the crystalline form ofTETA.4HCl has a Raman spectrum having shifts at 943 and 1173±5 cm⁻¹,most preferably 943 and 1173±2 cm⁻¹. Typically, the Raman spectrum issimilar to that shown in FIG. 5a (upper spectrum).

Raman spectra can, for example, be obtained using a Renishaw RA802Pharmaceutical Analyser. This can be operated at a laser wavelength of785 nm. Further operating conditions are set out in the Examples sectionherein.

The TETA.4HCl Form B crystalline form is storage stable. Thus,typically, the XRPD pattern and/or the Raman spectrum of a sample of thecrystalline form of the invention which has been stored at 20° C. for 6months, preferably 10 months, more preferably 12 months is identical, orsubstantially identical, to that of the crystalline form of theinvention described above. Preferably, at least 90 wt %, more preferablyat least 95 wt %, more preferably at least 98 wt % of a sample of thecrystalline form of the invention which has been stored at 20° C. for 6months, preferably 10 months, more preferably 12 months retains thecrystalline form, Form B, described herein.

The TETA.4HCl Form B crystalline form is stable in humid environments.Thus, typically, the XRPD pattern and/or the Raman spectrum of a sampleof the crystalline form of the invention which has been stored at 40° C.and 75% humidity for 1 month, preferably for four months, morepreferably for six months, is identical, or substantially identical, tothat of the crystalline form of the invention described above.Preferably, at least 90 wt %, more preferably at least 95 wt %, morepreferably at least 98 wt % of a sample of the crystalline form of theinvention which has been stored at 40° C. and 75% humidity for 1 monthretains the crystalline form, Form B, described herein. Preferably, atleast 90 wt %, more preferably at least 95 wt %, more preferably atleast 98 wt % of a sample of the crystalline form of the invention whichhas been stored at 40° C. and 75% humidity for 4 months, preferably for6 months, retains the crystalline form, Form B, described herein.

Preferably, the storage stability of the crystalline form of theinvention is determined by the XRPD pattern. Thus, preferably the XRPDpattern of a sample of the crystalline form of the invention which hasbeen stored at 20° C. for 6 months, preferably 10 months, morepreferably 12 months is identical, or substantially identical, to thatof the crystalline form of the invention described above. Preferably, atleast 90 wt %, more preferably at least 95 wt %, more preferably atleast 98 wt % of a sample of the crystalline form of the invention whichhas been stored at 20° C. for 6 months, preferably 10 months, morepreferably 12 months retains an identical or substantially identicalXRPD pattern to that of the crystalline form, Form B, described herein.Further, preferably the XRPD pattern of a sample of the crystalline formof the invention which has been stored at 40° C. and 75% humidity for 1month, preferably 4 months, more preferably 6 months, is identical, orsubstantially identical, to that of the crystalline form of theinvention described above. Preferably, at least 90 wt %, more preferablyat least 95 wt %, more preferably at least 98 wt % of a sample of thecrystalline form of the invention which has been stored at 40° C. and75% humidity for 1 month, preferably 4 months, more preferably 6 months,retains an identical or substantially identical XRPD pattern to that ofthe crystalline form, Form B, described herein.

Alternatively, the storage stability of the crystalline form of theinvention is determined by the Raman spectrum. Thus, preferably theRaman spectrum of a sample of the crystalline form of the inventionwhich has been stored at 20° C. for 6 months, preferably 10 months, morepreferably 12 months is identical, or substantially identical, to thatof the crystalline form of the invention described above. Preferably, atleast 90 wt %, more preferably at least 95 wt %, more preferably atleast 98 wt % of a sample of the crystalline form of the invention whichhas been stored at 20° C. for 6 months, preferably 10 months, morepreferably 12 months retains an identical or substantially identicalXRPD pattern to that of the crystalline form, Form B, described herein.Further, preferably the Raman spectrum of a sample of the crystallineform of the invention which has been stored at 40° C. and 75% humidityfor 1 month, preferably 4 months, more preferably 6 months, isidentical, or substantially identical, to that of the crystalline formof the invention described above. Preferably, at least 90 wt %, morepreferably at least 95 wt %, more preferably at least 98 wt % of asample of the crystalline form of the invention which has been stored at40° C. and 75% humidity for 1 month, preferably 4 months, morepreferably 6 months, retains an identical or substantially identicalRaman spectrum to that of the crystalline form, Form B, describedherein.

Particular advantages of the crystalline form of the invention relate toits storage stability. Storage of tablets obtained from Form A TETA.4HClare observed to have discoloured patches after storage for six months at40° C. and 75% humidity. A tablet obtained from TETA.4HCl Form A whichhas been aged is depicted in FIG. 7b . This shows the discolouration ofthe tablet over time. The present invention and the provision ofTETA.4HC Form B, in particular substantially pure TETA.4HCl Form B, isaimed at addressing this issue. Tablets obtained from TETA.4HCl Form Bare believed to have a reduced tendency to discolour over time.

The crystalline form of the invention typically has an FTIR spectrumhaving peaks at two or more, preferably four or more, more preferablyfive or six or more, most preferably all, of 1475, 1525, 16010, 2380,2435, 2580, 2830 and 2880±5 cm⁻¹. Preferably, the crystalline form ofthe invention has an FTIR spectrum having peaks at 1525, 2435 and 2675±5cm⁻¹, most preferably at 1526, 2436 and 2674±2 cm⁻¹. Preferably, thecrystalline form of the invention contains no more than 50 wt %, e.g. nomore than 40 wt %, preferably no more than 20 wt %, more preferably nomore than 10 wt % of a crystalline form having a peak at 943±2 cm⁻¹ inthe FTIR spectrum. Most preferably, the crystalline form issubstantially free of a crystalline form having a peak at 943±2 cm⁻¹.

FTIR spectra are typically FTIR-ATR spectra and can be obtained using aNicolet iS5 FT-IR spectrometer in ATR diamond mode. Specific conditionssuitable for obtaining FTIR spectra are set out in further detail inExample 4.

The crystalline form of the invention typically has a meltingtemperature of about 260° C., typically about 259° C. as measured byDSC. DSC analysis can be performed as set out in Example 4. For example,analysis can be performed using a Toledo DSC3+ device and providingsamples in a 40 μL sealed aluminium pan with the lid punctured beforeanalysis, under nitrogen flush, a 50 mL/min.

Analysis of the crystalline form of the invention by DVS can also beused to distinguish the present crystalline Form B from Form A. Thecrystalline form of the invention typically shows a weight gain at 90%RH and above of from 50-59%, typically from 54-57%. Typically, aftercompletion of a sorption and desorption cycle (0% to 95% RH) the weightgain of the sample is no more than 10%, preferably no more than 5%. Thiscontrasts with TETA.4HCl Form A which shows a weight gain followingsorption/desorption (0-95% RH) of 14-15%.

Typically, the crystalline form of TETA.4HCl according to the inventioncontains no more than 50 wt %, e.g. no more than 40 wt %, preferably nomore than 20 wt % more preferably no more than 10 wt % TETA.4HCl Form A.Preferred crystalline forms of TETA.4HCl according to the invention aresubstantially free of TETA.4HCl Form A. Substantially free of Form A asused herein means that the crystalline form contains no more than 5 wt %Form A, preferably no more than 2 wt %, more preferably no more than 1wt % and most preferably no more than 0.5 wt % or no more than 0.1 wt %Form A.

TETA.4HCl Form A is the crystalline form obtained under standardcrystallisation conditions, such as those described in Reference Example3 herein. Form A is characterised by an XRPD pattern having peaks at25.2 and 35.7±0.1°2θ, typically at 25.2 and 35.7±0.05°2θ. Preferably theXRPD spectrum of Form A also has peaks at 21.8, 26.9 and 28.2 0.1°2θ,typically at 21.8, 26.9 and 28.2±0.05°2θ. In particular Form A ischaracterised by an XRPD pattern as set out in FIG. 3 (“Profile 1”).Form A may also be characterised by a Raman spectrum having peaks at 933and/or 1513±5 cm⁻¹, typically at 933 and/or 1513±2 cm⁻¹. In particular,Form A is characterised by a Raman spectrum having peaks at 933, 1167,1513 and 1604±5 cm⁻¹, typically at 933, 1167, 1513 and 1604±2 cm⁻¹.Typically, Form A is characterised by a Raman spectrum as set out inFIG. 5a (lower spectrum) herein.

Preferably, the crystalline form according to the invention contains nomore than 50 wt %, e.g. no more than 40 wt %, preferably no more than 20wt % more preferably no more than 10 wt %, more preferably no more than5 wt %, no more than 2 wt %, no more than 1 wt % and most preferably nomore than 0.5 wt % or no more than 0.1 wt % of a crystalline form ofTETA.4HCl having an XRPD pattern having peaks at 25.2 and 35.7±0.1°2θ,or having peaks at 21.8, 25.2, 26.9, 28.2 and 35.7±0.1°2θ.

Preferably, the crystalline form according to the invention contains nomore than 50 wt %, e.g. no more than 40 wt %, preferably no more than 20wt % more preferably no more than 10 wt %, more preferably no more than5 wt %, no more than 2 wt %, no more than 1 wt % and most preferably nomore than 0.5 wt % or no more than 0.1 wt % of a crystalline form ofTETA.4HCl having a Raman spectrum having peaks at 933 and/or 1513±5cm⁻¹, typically at 933 and/or 1513±2 cm⁻¹, or having peaks at 933, 1167,1513 and 1604 cm⁻¹±5 cm⁻¹, typically at 933, 1167, 1513 and 1604±2 cm⁻¹.

Preferably, the crystalline form of TETA.4HCl contains at least 90 wt %Form B. More preferably, the crystalline form consists essentially ofForm B, i.e. it is substantially pure TETA.4HCl Form B. Where acrystalline form consists essentially of Form B, it typically containsat least 95 wt % TETA.4HCl Form B, more preferably at least 98 wt %,more preferably at least 99 wt %, and most preferably at least 99.5 wt %or 99.9 wt % TETA.4HC Form B, wherein TETA.3HCl Form B is characterisedby an XRPD spectrum and/or a Raman spectrum as set out herein,preferably TETA.4HCl Form B is characterised by an XRPD spectrum as setout herein.

The TETA.4HCl crystals described herein are typically provided in driedform. Thus, they typically contain less than 1 wt % water, preferablyless than 0.5 wt % water, more preferably less than 0.1 wt % or 0.05 wt% water. Total residual solvent is preferably less than 0.1 wt %, morepreferably less than 0.5 wt %.

Methods of Manufacturing Crystalline TETA.4HCl

TETA.4HCl can be produced by techniques known in the art. For example,TETA free base is commercially available and can be converted to thecrystalline TETA hydrate and isolated by routine methods. The TETAhydrate can be treated with aqueous HCl to provide the TETA.4HCl salt.Typically, the TETA.4HCl salt is isolated in crude form beforerecrystallization as the Form B polymorphic form.

TETA.4HCl in crystalline form can be obtained by an anti-solventcrystallisation process, typically from the aqueous solution. Suchprocess involves addition of an anti-solvent to an aqueous solution ofTETA.4HCl and collecting the resulting crystals. When carried out understandard crystallisation conditions, for example by crystallising atroom temperature or above, and/or by a method including drying atelevated temperature, such methods have been found to lead to a singlecrystalline form of TETA.4HC, known herein as Form A. Form A crystalswere obtained even on variation of the solvent system.

For instance, the present inventors have produced TETA.4HCl using themethods described in WO 2006/027705, and found that these methods leadto production of Form A crystals. The inventors reproduced Example 17 ofWO 2006/027705, starting from a mixture of isomers oftriethylenetetramine, and using the crystallisation conditions asdescribed in Example 17 of WO 2006/027705. The product was analysed byXRPD and the results are set out in FIGS. 12a to 12c . The productobtained contained the characteristic peaks of TETA.4HCl Form A.However, certain peaks known to be characteristic of TETA.4HC Form Bwere absent, in particular those at around 35° 2θ and that at 25.4° 2θ,suggesting that the product produced was TETA.4HCl Form A, the formwhich is known to be produced by standard room temperaturecrystallisation.

The present inventors have found that, using the same solvent system butvarying the crystallisation conditions, in particular the time andtemperature of processing, Form B crystals can be obtained.

At temperatures of about 20° C. or below, in particular about 15° C. orbelow, TETA.4HCl may be produced as Form B. From about 20° C. to 30° C.,the crystalline form produced may be dependent on conditions other thansimply the temperature of crystallisation. Thus, above about 20° C.further conditions also typically need to be controlled in order toensure that Form B is produced. In particular, the crystalline formproduced may be dependent on the rate of crystallisation. Thus, a slowcrystallisation favours formation of Form B, whereas more rapidcrystallisation favours Form A. Even at temperatures of from 15-20° C.,some Form A crystals may be produced unless crystallisation is carriedout slowly. For example where anti-solvent addition is used to formcrystals, anti-solvent should preferably be added slowly to the solutionin order to ensure that Form B, rather than Form A, is produced.

Typically, the crystalline form of the invention is produced bycrystallisation at a temperature of about 20° C. or below, preferablyabout 15° C. or below, more preferably about 10° C. or below. In oneembodiment, preferred temperatures for the crystallisation are 13° C. orbelow, more preferably from 7-13° C. At temperatures of about 15° C. orbelow, particularly at 13° C. or below, Form B is the thermodynamicallyfavoured form and crystallisation will generally result in substantiallypure Form B.

Preferably, all steps in the crystallisation process are carried outbelow 30° C., preferably about 20° C. or below, preferably about 15° C.or below, more preferably about 10° C. or below. Where the temperatureis above about 15° C., a mixture of Form A and Form B may be produced.Where the temperature is above about 30° C., only Form A will result. Toensure that the product produced is substantially pure Form B, thetemperature is preferably kept at about 15° C. or below at all timesduring crystallisation. At temperatures between about 15° C. and 20° C.,Form B crystals can be produced by carrying out crystallisation at aslow rate of anti-solvent addition. In particular, addition of Form Bseed crystals combined with slow solvent addition encourages formationof substantially pure Form B. Addition of anti-solvent in a slow andcontrolled fashion ensures that crystallisation develops from the seedcrystal and separate nucleation of Form A does not occur.

Typically, crystallisation is carried out by anti-solvent addition at arate of 0.5 ml/min or less of anti-solvent added to an aqueous solutionof TETA.4HC, per gram of TETA.4HC dissolved in the aqueous solution.Thus, the preferred rate of addition is 0.5 ml anti-solvent, per minute,per gram of TETA.4HCl or less, i.e. 0.5 ml/min/g or less. Preferredrates of anti-solvent addition are 0.2 ml/min/g or less, more preferablyabout 0.1 ml/min/g or less. Preferred rates of addition are from 0.01 to0.2 ml/min/g, most preferably from 0.01 to 0.1 ml/min/g.

Thus, to provide substantially pure Form B crystals, crystallisation ispreferably carried out at about 15° C. or below and preferably at a rateof addition of 5 ml/min/g or less, more preferably 0.2 ml/min/g or lessfor example about 0.1 ml/min/g. Most preferably crystallisation iscarried out at 13° C. or below, e.g. from 7 to 13° C., and preferably ata rate of addition of less than 0.2 ml/min/g, for example about 0.1ml/min/g or less.

Seed crystals of TETA.4HCl Form B are preferably added. Seed crystalsmay be added either before, during or after anti-solvent addition,typically either before or during anti-solvent addition, most preferablybefore anti-solvent addition. If seed crystals are added either duringor after anti-solvent addition, they are preferably added before theformation of crystals are observed.

A preferred method of crystallisation uses TETA.4HC, preferably purifiedTETA.4HC, as a starting material. The presence of impurities in thestarting material can impact the ability to crystallise the desiredpolymorph. Thus, TETA.4HCl is preferably in isolated form, i.e. it isisolated from any reaction mixture in which it is was produced (andtypically purified) before crystallisation to produce Form B iscommenced. Further, the crude TETA.4HCl is preferably recrystallizedbefore the process to produce Form B is commenced. This also provides ahigher purity starting material and enables Form B crystals reliably tobe produced by following the methods as set out herein.

TETA.4HCl is typically dissolved in aqueous solution prior tocrystallisation. Typically, the solution comprises from 0.01 to 10 gTETA.4HCl per ml of water. Preferably, the solution comprises from 0.1to 5 g TETA.4HCl per ml of water, most preferably from 0.6 to 1.2 gTETA.4HCl per ml of water. The volume of anti-solvent used for therecrystallization is typically 0.5 ml or less, per gram of TETA.4HCldissolved in the aqueous solution. Preferred amounts of anti-solvent are0.2 ml/g TETA.4HCl or less, more preferably about 0.1 ml/g TETA.4HCl orless. Preferred amounts of anti-solvent are from 0.01 to 0.2 ml/gTETA.4HC, most preferably from 0.01 to 0.1 ml/g TETA.4HCl.

Preferred methods of crystallisation of Form B comprise:

-   (i) Adding an anti-solvent to an aqueous solution, preferably an    agitated aqueous solution, of TETA.4HCl at temperature T1 over a    period of time t1 and/or at a rate of addition R1;-   (ii) Optionally adding TETA.4HCl seed crystals;-   (iii) Optionally agitating the resulting mixture at T1 for a further    period tla;-   (iv) Optionally reducing the temperature to temperature T2 and    agitating the mixture for a further period t2; and-   (v) Collecting the resulting crystals.

T1 is about 20° C. or below, preferably about 15° C. or below, morepreferably about 10° C. or below. In order to produce substantially pureForm B, T1 is preferably about 15° C. or below, more preferably about10° C. or below.

The anti-solvent may be any solvent in which the TETA.4HCl issubstantially insoluble. Suitable anti-solvents include ethanol,methanol, acetonitrile, propan-2-ol, acetone and 1,4-dioxane andmixtures thereof. Methanol and ethanol and mixtures thereof arepreferred, in particular ethanol.

The solution is typically agitated or mixed during addition, typicallyby stirring. Further agitation of the mixture, e.g. stirring, may beapplied during steps (iii) and (iv).

The crystallisation process is typically carried out over an extendedperiod of time. Thus, t1 is typically at least 1 hour, preferably atleast 1.5 hours. The rate of addition of anti-solvent R1 is typically0.5 ml/min/g or less. Preferred rates R1 are 0.2 ml/min/g or less, morepreferably about 0.1 ml/min/g or less, e.g. from 0.01 to 0.2 ml/min/g,most preferably from 0.01 to 0.1 ml/min/g.

If seed crystals are added, these are typically added before, during orafter step (i) is carried out. Preferably, seed crystals are addedeither before step (i) or during the anti-solvent addition of step (i).If seed crystals are added during or after addition of anti-solvent, themixture is typically stirred for a further prolonged period, tla, whichis preferably at least 2 hours, e.g. at least 3 hours or 4 hours, forexample about 5 hours. Preferably, a further stirring period at a lowertemperature is also included prior to collection of crystals. Thisfurther stirring step is carried out for a period t2 which is preferablyat least 30 minutes. The further stirring step is carried out at reducedtemperature, T2. T2 is typically less than T1, preferably at least 5°C., more preferably at least 10° C. less than T. T2 is typically about10° C. or below, preferably about 5° C. or below, more preferably about0° C. Increasing the time or rate of crystallisation and reducing thetemperature of crystallisation has been found to provide greater purityof Form B. Introducing seed crystals also helps to improve the purity ofthe Form B crystalline form.

In the method above, typically, T1 is about 20° C. or below; T2 is atleast 5° C. lower than T1; t1 is at least 1 hour, R1 is 0.5 ml/min/g orless, t1 a is at least 2 hours and t2 is at least 30 minutes. PreferablyT1 is about 20° C. or below; T2 is about 10° C. or below; t1 is at least1 hour, R1 is 0.5 ml/min/g or less, t1 a is at least 3 hours and t2 isat least 30 minutes. More preferably T1 is about 15° C. or below; T2 isabout 5° C. or below; t1 is at least 1 hour, R1 is 0.2 ml/min/g or less,t1 a is at least 4 hours and t2 is at least 30 minutes.

Crystallisation is preferably carried out under inert atmosphere, forexample under nitrogen.

Preferred crystallisation methods include at least steps (i), (iv) and(v) above. More preferred methods include steps (i), (iii), (iv) and(v). Most preferred methods include all of steps (i) to (v). Thus, apreferred method of manufacturing TETA.4HCl comprises:

-   (i) adding anti-solvent to an aqueous solution, typically an    agitated aqueous solution, of TETA.4HCl at temperature T1 over a    period of time t1 and or at a rate R1;-   (ii) optionally adding TETA.4HCl seed crystals;-   (iii) optionally agitating the resulting mixture at T1 for a further    period tla;-   (iv) reducing the temperature to temperature T2 and agitating the    mixture for a further period t2; and-   (v) collecting the resulting crystals;    wherein T1 is about 20° C. or below; T2 is about 10° C. or below; t1    is at least 1 hour, R1 is 0.5 ml/min/g or less, t1 a is at least 3    hours and t2 is at least 30 minutes.

A further preferred method, which is suitable for producingsubstantially pure TETA.4HCl Form B comprises:

-   (i) adding anti-solvent to an aqueous solution, typically an    agitated aqueous solution, of TETA.4HCl at temperature T1 over a    period of time t1 and/or at a rate R1;-   (ii) adding TETA.4HCl seed crystals;-   (iii) agitating the resulting mixture at T1 for a further period    tla;-   (iv) reducing the temperature to temperature T2 and agitating the    mixture for a further period t2; and-   (v) collecting the resulting crystals;    wherein T1 is about 15° C. or below; T2 is about 5° C. or below; t1    is at least 1 hour, R1 is 0.2 ml/min/g or less, t1 a is at least 4    hours and t2 is at least 30 minutes.

Crystals may be collected by any suitable means as long as thetemperature of the collection steps is maintained below about 40° C.,preferably below about 30° C. Higher temperature steps carried outbefore the crystals have been fully dried have been found to lead toForm A crystals only. Most preferably, collection of the crystals iscarried out at below about 25° C., for example about 20° C. or below.

Suitable methods for collecting crystals include filtration andcentrifuging. Typically, the resulting crystals are then dried,typically at a temperature of below about 40° C., preferably below about30° C. Crystals may be washed, for example with anti-solvent, prior todrying. Suitable anti-solvents for washing are those mentioned above, inparticular methanol or ethanol, most preferably ethanol. Drying istypically vacuum drying, since heating will lead to Form A crystalsbeing produced. Vacuum drying at less than about 40° C. is preferred.

The resulting dried product may be further processed, for example bymilling or granulation, if desired. Crystal Form B is substantiallystable on milling.

Where relevant, collection and further processing steps such as washing,drying and milling are typically carried out under inert atmosphere,such as under nitrogen.

Pharmaceutical Compositions and Dosage Forms

The pharmaceutical compositions of the invention comprise crystallineTETA.4HC Form B as described herein together with one or morepharmaceutically acceptable carriers or diluents. The pharmaceuticalcomposition may take any suitable form, but is preferably an oral dosageform. For example, the composition may take the form of a tablet, acapsule, a powder, a semisolid, a sustained release formulation, asolution, a suspension or any other appropriate composition. Tablets,capsules and powders, in particular tablets, are preferred.

In alternative embodiments, the compositions are administeredparenterally, for example subcutaneously or intravenously.

The pharmaceutical dosage form may be produced by carrying out furtherprocessing steps on the crystals produced as described herein. Thus, acomposition, typically an oral dosage form, may be produced by (a)obtaining TETA.4HCl Form B, for example using the method describedabove, (b) optionally milling and/or granulating the crystals obtained,(c) combining the TETA.4HCl Form B with a pharmaceutically acceptablecarrier, and (d) optionally mixing the TETA.4HCl Form B and the carrier.Suitable carriers are described further below. Where the oral dosageform is a tablet, the process may further comprise (e) compressing themixture to form a tablet and optionally sugar-coating or film-coatingthe tablet. Alternatively, the solid oral dosage form may be a capsuleor a powder. In this case, the method of the invention may furthercomprise (e) packaging the resulting mixture, for example in a capsule.Further standard steps may be included in the process, for examplemilling, granulating, sugar-coating, or film coating.

The pharmaceutical composition typically comprises up to 85 wt % ofTETA.4HCl, for example up to 50 wt % TETA.4HCl. Preferred compositionsare sterile and pyrogen free.

Suitable pharmaceutically acceptable carriers for the preparation oforal dosage forms include, for example, solubilising agents, e.g.cyclodextrins or modified cyclodextrins; diluents, e.g. lactose,dextrose, saccharose, cellulose, corn starch or potato starch;lubricants, e.g. silica, talc, stearic acid, magnesium or calciumstearate, and/or polyethylene glycols; binding agents; e.g. starches,tragacanth gums, gelatin, syrup, acacia, sorbitol, methylcellulose,carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents,e.g. starch, alginic acid, alginates or sodium starch glycolate;effervescing mixtures; dyestuffs; sweeteners; wetting agents, such aslecithin, polysorbates, laurylsulphates; and, in general, non-toxic andpharmacologically inactive substances used in pharmaceuticalformulations. Such pharmaceutical preparations may be manufactured in aknown manner, for example, by means of mixing,

The compositions of the invention typically contain a high proportion ofTETA.4HC Form B and a low amount of Form A. Preferably, thepharmaceutical compositions of the invention contain no more than 10 wt% TETA.4HCl Form A. Preferably, the compositions of the invention aresubstantially free of triethylenetetramine tetrachloride Form A.Substantially free of Form A as used herein means that the compositioncontains no more than 5 wt %, preferably no more than 2 wt %, morepreferably no more than 1 wt %, or 0.5 wt %, and most preferably no morethan 0.1 wt % TETA.4HCl Form A.

Medical Uses

A therapeutically effective amount of a compound of the invention isadministered to a subject. It will be understood that the specific doselevel for any particular subject will depend upon a variety of factorsincluding the activity of the specific compound employed, the age, bodyweight, general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination and the severity ofthe particular disease undergoing treatment. Optimum dose levels andfrequency of dosing will usually be determined by clinical trial.

A typical daily dose is up to 50 mg per kg of body weight, for examplefrom 0.001 to 50 mg per kg of body weight, according to the age, weightand conditions of the subject to be treated, the type and severity ofthe disease and the frequency and route of administration. Preferably,daily dosage levels are from 0.05 mg to 2 g, preferably from 0.1 mg to10 mg. The compound of the invention is typically administered to thepatient in a non-toxic amount.

The invention also provides a crystalline form as defined herein or acomposition as defined herein for use in a method of treatment of thehuman or animal body by therapy. In particular the crystalline forms andcompositions of the invention are useful in reducing copper levels in asubject and/or reducing the toxic effects of copper retention in asubject. They are therefore useful in the treatment of disorders anddiseases associated with raised copper levels. In particular, they areuseful in the prevention or treatment of Wilson's disease.

TETA itself is a known treatment for Wilson's disease and diseases anddisorders associated with elevated copper levels. After administrationof the crystalline form of the invention, the compounds will bedissolved in the in vivo system and the therapeutic effect of thecrystalline form can be expected to be the same as known forms of TETA.

The subjects treated according to the present invention may be human oranimal subjects, in particular humans or mammals, typically humans.

EXAMPLES Reference Example 1: Synthesis of TETA.4HCl Crude Form

TETA.4HCl in crude form was produced as set out in FIGS. 1a and 1 b.

Example 2: Synthesis of TETA.4HCl Form B

TETA.4HCl Form B in substantially pure form was produced by followingthe steps set out in FIG. 2.

Reference Example 3: Synthesis of TETA.4HCl Form A

Crude TETA 4HCl was dissolved under nitrogen in 2 volumes of purifiedwater, and then the solution obtained was clarified by filtration. Areactor was heated at 70° C. (jacket reactor temperature) and when thetemperature of the reaction mixture was between 55 and 60° C., 7 volumesof methanol were added to recrystallize the product, at a rate such thatthe temperature in the mass remained between 55 and 65° C.

After at least 30 minutes of stirring at 65° C. (jacket reactortemperature), the reaction mixture was cooled slowly over a period of atleast 2 hours 30 min, while respecting a cooling speed of around 5° C.per 30 min, to obtain a temperature in the mass between 30° C. and 35°C. The suspension was then stirred at least for 1 hour at a temperaturein the mass maintained between 30° C. and 35° C.

The product was filtered on an enameled Nutsch filter with 10 μmmembrane porosity and washed twice with 1.5 volumes of methanol. Forcedfiltration was performed after the last wash to dry the product. Theproduct was dried in a vacuum oven at 60° C. for at least 14 hours.

Example 4: Analysis of TETA.4HCl Crystal Forms

X-Ray Powder Diffraction

A few milligrams of the samples obtained in Example 2 and ReferenceExample 3 above was placed between three polymer foils (Kapton® andpolypropylene). Kapton® exhibits a broad peak in the diffractogram witha weak intensity around 2θ=5.5°.

Samples were placed in a PANALYTICAL X'PERT PRO MPD diffractometerconfigured in transmission mode, and analysed using conditions indicatedin Table 1 below. Diffraction data is acquired by exposing powdersamples to Cu—K_(α) X-ray radiation, which has a characteristicwavelength (λ) of 1.5418 Å. X-rays were generated from a Cu anodesupplied with 40 kV and a current of 40 mA. The analyses were performedbetween 2° and 50° (unless stated otherwise). The calibration of thediffractometer was validated before each analysis.

FIG. 3 shows an XRPD pattern for a mixture of Form A and Form B(“Profiles 1+2”) as well as an XRPD pattern for crystals produced inaccordance with Reference Example 3 (substantially pure Form A: “Profile1”). Arrows mark the peaks unique to Form B. FIG. 4 shows an XRPDpattern for crystals produced in accordance with Example 2(substantially pure Form B).

TABLE 1 XRPD Analysis Conditions Type X'Pert Pro MPD Panalytical SerialNumber DY2764 Incident Beam Radius (mm) 240.0 (Transmission Mode) X-raytube: Name PW3373/10 Anode Material: Cu Voltage (kV): 40 Current (mA):40 Focus type: Line (length (mm): 12.0 width (mm): 0.4 Take-off angle(°): 4.4) X-ray Mirror: Name: Inc. Beam Cu W/Si (parabolic MPD) Crystal(W/Si Graded Parabolic) Acceptance angle (°): 0.8 Length (mm): 55.3Soller Slit Soller 0.04 rad. Opening (rad.): 0.04 Anti-scatter slit: ASSlit 1.4 mm (mirror) Type: Fixed Height (mm): 1.40 Divergence slit SlitFixed 1/8° Distance to Sample (mm): 140 Type: Fixed Height (mm): 0.19Diffracted Beam Radius (mm): 240.0 Soller slit Name: Large Soller 0.04rad. Opening (rad.): 0.04 Detector Name: PIXcel Type: RTMS detectorPHD - Lower level (%): 25.5 PHD - Upper level (%): 70.0 Mode: ScanningActive length (°): 3.347Raman Spectroscopy

Samples of Example 2 and Reference Example 3 were analysed by Ramanspectroscopy. A Renishaw RA802 Pharmaceutical Analyser was used underthe following conditions:

TABLE 2 Laser wavelength 785 nm Spectral dispersion 2 cm⁻¹/pixelObjective Hi Mag (×50 − 1 μm capability) Focussing Automatic (LiveTrack)Acquisition time 1 s Laser power 50%

The spectra are provided in FIGS. 5a and 5b . In FIG. 5b , the lowerline represents Example 2 (Form B), whilst the upper line representsReference Example 3 (Form A). Arrows depict shifts unique to Form B. TheRaman spectrum for Example 2 shows bands at 943, 1173, 1527 and 1612cm⁻¹. The Raman spectrum for Reference Example 3 (Form A) shows peaks at933, 1167, 1513 and 1604 cm⁻¹.

FTIR-ATR Analysis

Infrared spectra are measured on a Nicolet iS5 FT-IR spectrometerequipped with an iS7 ATR module, with the parameters set out below:

TABLE 3 Mode ATR Diamond Resolution 4 cm⁻¹ Number of scans 32 scans(measurement) Number of scans 32 scans (background) Spectrum 4000 cm⁻¹to 525 cm⁻¹, in absorbance

An infra-red spectrum was obtained for Example 2 (Form B). The spectrumis shown in FIGS. 8a and b.

DSC Analysis

DSC analyses were performed using a Mettler Toledo DSC3+(serial numberB531255222) in 40 μl sealed aluminium pans with the lid punctured beforeanalysis, under nitrogen flush at 50 mL/min.

TABLE 4 Sample ID Example 2 Sample weight 0.360 mg Scanning range 20°C.-300° C. Scanning rate 10°/min

An endothermal event corresponding to the melting of the sample isobserved. Onset and peak temperatures are shown in the Table below.

TABLE 5 T Onset (° C.) T Peak (° C.) Enthalpy (J/g) Comment 252.1 259.2232.1 Melting Onset, peak temperatures and enthalpies

Thermogravimetric Analysis (TGA)

Thermogravimetric analyses were performed using a Pyris 1 TGA analyser(serial number 537N7052501) in sealed aluminium pans, punctured beforeanalysis, under nitrogen flush at 20 mL/min.

TABLE 6 Sample ID Example 2 Sample weight 5.100 mg Start temperature 25°C. End temperature 300° C. Scanning rate 10° C./min

The thermogravimetric analysis shows a weight loss starting at 225° C.,which increases after 289.5° C. This is likely due to degradation. Weighloss was 10.24%: 2.64% between 225.0-289.5° C. and 7.60% between289.5-299.0° C.

Dynamic Vapour Sorption (DVS) Analysis

DVS analyses are performed using an SMS DVS Intrinsic analyser (serialnumber PF 140088) in open aluminium pans at 25° C. with a nitrogen purgegas at 100 ml/min. The stability criterion was a weight change lowerthan 0.002% on a 5 minute time frame (with a minimum of 10 min and amaximum of 100 min).

TABLE 7 Sample ID Example 2 Sample 11.2773 mg (initial) weight 11.2763mg (ref. 0% RH) Temperature 25° C. Relative 1) 40% RH-0% RH Humidity 2)0% RH-95% RH program 3) 95% RH-0% RH

DVS Isotherm plots are provided in FIGS. 11a (Example 2) and 11 b (RefExample 3). The DVS analyses performed on these two samples show asignificant weight gain at high relative humidity values (90% RH andabove):

-   -   at least +55.8% for Example 2    -   at least +60.8% for Ref Example 3

The desorption stages exhibit different behaviours for the two solids.Example 2 almost reaches its reference weight on the second desorptionstage. For Ref Example 3 the weight is still +14.4% higher than thereference weight at the end of the second desorption stage (still +7.9%higher for the minimum value reached). It is worth noting that for thelatter the time limit criterion was reached on these steps (the weightof the sample is therefore not stabilized).

Example 5: Heckel Test

The aim of the Heckel test is to compress a test material undercontrolled conditions to derive the yield pressure of the bulk material.A known weight of material is compressed within a 10 mm diameter diewith flat faced punches moving at a set speed. The force on the punch isaccurately measured at frequent intervals whilst the displacement of thepunches is used to calculate the volume of the powder. The yieldpressure is calculated at slow and fast punch speeds to assess the timedependant component to deformation of the material. Samples producedaccording to Example 2 and Reference Example 3 were subjected to theHeckel test.

Methodology

Determination of True Density by Helium Pychnometry.

Equipment used: Micromeritics AccuPyc 111340

Test Parameters:

Cup size 3.5 cm3

Number of Purges 5

Purge pressure 19.5 psig

Number of runs 10

Run fill pressure 19.5 psig

Equilibration rate 0.02 psig

Run Precision Yes

Percentage full scale 0.05%

Testing was performed in duplicate. (Assuming target <2% variabilityachieved).

Compression

A known weight of pure drug is compacted to theoretical zero porosityusing 10 mm diameter flat faced punches. The Compaction Simulator wasused under the following conditions:

Tooling: 10 mm round flat faced

Profile: V shaped profile

Punch speed—Slow 0.1 mm/s

Punch speed—Fast 300 mm/s

Lubrication of die: Yes with Mg stearate in acetone

Number of repeats: 3

Elasticity correction: Yes

During compression the location of the punch tips are accuratelydetermined and the force measured by load cells producing a record ofthe primary compression parameters. Temperature and humidity weremonitored at intervals during the test.

The data were analysed by the Compaction Analysis software programme togenerate values for yield pressure (Py) using the Heckel equation:

${\ln\frac{1}{1 - D}} = {{kP} + A}$where D=the relative density of the compactP=Pressure appliedK=Gradient of the line in the linear region[Reference: R. W. Heckel. Trans. Metall. Soc. AIME 221 (1961)1001-1008]Strain Rate Sensitivity (SRS)For some materials, the deformation characteristics change with rate ofapplied force. This can be estimated by calculating the Strain RateSensitivity. The yield pressure at high speed compression is compared tothat at slow speed using the following equation:

${\%\mspace{14mu}{SRS}} = {\frac{{{Py}\mspace{14mu}{Fast}} - {{Py}\mspace{14mu}{Slow}}}{{Py}\mspace{14mu}{Slow}} \times 100}$[Reference: R. J. Roberts and R. C. Roe, Chem. Eng. Sci. 42(1987) p903].ResultsTrue Density

TABLE 8 Run Ref Example 3 Example 2 Run 1 1.3523 g/cm³ 1.3693 g/cm³ Run2 1.3431 g/cm³  1.3973 g/cm³* Run 3 1.3678 g/cm³ MEAN 1.3477 g/cm³1.3686 g/cm³ (SD 0.013) (SD 0.002) *Variation between runs 1&2 exceeds2%. 3^(rd) run performed. Data from run 2 assumed an outlier and notused in the mean calculation.

TABLE 9 Compaction Results: Ref Example 3 Slow speed 0.1 mm/s Run 1 Run2 Run 3 Yield Pressure 101.529 95.501 97.474 (Mpa) Range of linear25-150 25-150 25-150 region used in calculation (Mpa) Peak Force of20.830 18.543 19.570 Upper punch (kN) Ejection Force 0.039 −0.007 −0.020(kN) Compact Good Good Good observations: shiny shiny shiny tablettablet tablet Compact strength 6.41 7.45 11.17 (Kiloponds) Labconditions: 21.3° C./52.1% RH

TABLE 10 Compaction Results: Ref Example 3 Fast Speed 300 mm/s Run 1 Run2 Run 3 Yield Pressure 127.197 125.309 129.748 (Mpa) Range of linear25-150 25-150 25-150 region used in calculation (Mpa) Peak Force of14.644 14.752 14.617 Upper punch (kN) Ejection Force 0.421 0.327 0.381(kN) Compact Good but Good but Good but observations: blisteringblistering blistering on upper on upper on upper surface surface surfaceand and and chipping chipping chipping at edges at edges at edgesCompact strength 6.59 4.27 4.75 (Kiloponds) Lab conditions: 21.6°C./50.0% RH

TABLE 11 Summary of results and observations Property of Batch RefExample 3 Yield pressure  98.17 Mpa (±3.07) Slow (0.1 mm/s) Yieldpressure 127.42 Mpa (±2.23) fast (300 mm/s) Strain rate 29.8%sensitivity

TABLE 12 Compaction Results: Example 2 Slow speed 0.1 mm/s Run 1 Run 2Run 3 Run 4 Yield Pressure 106.465 117.516 118.116 115.227 (Mpa) Rangeof linear 25-150 25-150 25-150 25-150 region used in calculation (Mpa)Peak Force of 20.211 24.088 24.175 23.906 Upper punch (kN) EjectionForce 0.044 0.053 0.080 0.060 (kN) Compact Good Good Good Goodobservations: shiny shiny shiny shiny tablet tablet tablet tabletCompact strength 12.07 7.52 9.64 9.19 (Kiloponds) Lab conditions: 21.7°C./51.1% RH

TABLE 13 Compaction Results: Example 2 Fast Speed 300 mm/s Run 1 Run 2Run 3 Yield Pressure 129.865 129.108 129.861 (Mpa) Range of linear25-150 25-150 25-150 region used in calculation (Mpa) Peak Force of14.566 14.542 14.717 Upper punch (kN) Ejection Force 0.441 0.456 0.417(kN) Compact Good but Good Good observations: blistering shiny shiny toupper tablet. tablet. surface Sticking Sticking and lower and and punchchipping chipping sticking to lower to lower edge. edge. Compactstrength 9.83 11.58 8.88 (Kiloponds) Lab conditions: 21.7° C./51.2% RH

TABLE 14 Summary of results and observations Property of Batch Example 2Yield pressure 114.33 Mpa (±5.39) Slow (0.1 mm/s) Yield pressure 129.61Mpa (±0.44) fast (300 mm/s) Strain rate sensitivity 13.4%

The Heckel test is a measure of the deformation of a formed tablet. Thecompact strength is an indication of how the dwell time affects bondingof the compact. It was found that Reference Example 2 compacts producedat slow speed had moderate tensile strength (6-11 kiloponds). At fastspeed, strength reduced to about 4-6 kiloponds. Example 3 on the otherhand showed strengths of 7-12 kiloponds at slow speed, but 9-11kiloponds at fast speed, showing that Example 3 has a greater tensilestrength of compacted product at fast compaction fates. FIG. 6 shows thestrain rate sensitivity of Example 2 and Reference Example 3 atdifferent production speeds.

Example 6: Aging of Tablets

TETA.4HCl obtained in accordance with Reference Example 3 was compressedto form a tablet. An image of the tablet is provided in FIG. 7a . Thetablet was aged for six months at 40° C. and 75% humidity. After aging,the tablet was observed to have a number of discoloured patches. Animage of the aged tablet is provided in FIG. 7 b.

The tablet was analysed by Raman spectroscopy under the same conditionsas are set out in Example 4 above and the results compared with theRaman spectra for Form A and Form B TETA.4HC. FIG. 7c shows in darkenedregions the areas of the tablet which show the presence of TETA.4HClForm A. As is apparent from a comparison of FIGS. 7b and c , the areasof TETA.4HCl Form A correlate to the location of the discoloured regionson the tablet surface.

Minute amounts of TETA.4HCl Form B were detected in the tablet which mayhave formed under compression of TETA.4HCl to form a tablet. The areasof TETA.4HCl Form B do not correlate to the discoloured regions observedin the aged tablet.

Example 7: Crystallisation Process

Crystallization tests for the preparation of Form B were carried out at20° C. Starting solutions were prepared in a mixture of ethanol/water at(25:75) and the addition of ethanol was performed until a (75:25) ratiowas reached. First tests were carried out by manually adding ethanol.Additional tests were then performed using a syringe pump, for anaddition at a slow and controlled rate.

Manual Addition (Dropwise)

In these tests, the addition was carried out manually using amicropipette. A solution of TETA.4HCl in a (25:75) ethanol/water mixturewas placed under stirring and thermostated at 20° C. The amount ofTETA.4HCl starting material is set out in Table 15.

Anti-solvent (ethanol) was added dropwise, at a regular interval. Two“rates” of addition were tested. After the addition of the anti-solvent,the solid phase was sampled and analysed by X-ray diffraction in orderto determine the nature of the solid phases. Conditions of X-raydiffraction were as set out in Example 4. The last test was performedwith seeds of Form B present at the beginning of the addition, and at aslower rate of addition (PE1716E007-L-5). This test leads to a solidphase with no signal of Form A observed on the diffractogram.

The results are set out in Table 15 and the diffractograms obtainedafter the analysis of the solid phases in suspension at the end of theethanol addition are presented in FIGS. 9a, b and c . FIGS. 9a-c showXRPD diffractograms for, beginning at the lowest line:

Reference of Form A (Bottom Line in FIGS. 9a-c )

Reference of Form B

PE1716E007-L-5

PE1716E007-L-4

PE1716E007-L-3

PE1716E007-L-2

PE1716E007-L-1

PE1716E007-R-1 (top line in FIGS. 9a-c )

TABLE 15 Table 15. Results of the crystallization tests by manualaddition. m m Initial Final starting solvent Concentr. Concentr. Testmaterial mixture (mg/mg Seeds Solvent added Total (mg/mg XRD Profilereference (mg) (mg) solution) (mg) (μL) (mg) time solution) XRDreference PE1716E007-R-1 192.71 384.45 0.334 No 816 643.8 2 s 0.158 FormB + Form A PE1716E007-L-1 190.28 387.53 0.329 No 816 643.8 1 min 0.156Form B + Form A PE1716E007-L-2 702.48 1445.64 0.327 No 3000 2367 1 min0.156 Form B + Form A PE1716E007-L-3 703.91 1445.90 0.327 No 3000 2367 1min 0.156 Form B + Form A PE1716E007-L-4 748.33 1462.15 0.339 No 30002367 5 min 0.163 Form B + Form A (weak) PE1716E007-L-5 750 1452.53 0.3412.72 3000 2367 32 min 0.164 Form BProgrammed Addition (Continuous)

In this series of tests, the addition of the antisolvent was driven bysyringe pump. This allowed a continuous addition at a very low rate. Thesame protocol as above was used: a solution of the starting material wasprepared, close to saturation, in a 25:75 ethanol/water mixture as setout in Table 16. The solution was then saturated with Form B until asolid phase remained in suspension: this ensures the presence of seedsof Form B at the beginning of anti-solvent addition.

Two rates of addition were tested: 0.05 mL/min and 0.1 mL/min (for 750mg starting material in solution). Ethanol was added at these selectedrates until a 75:25 ethanol/water ratio was obtained. Two additionaltests were performed with an addition up to 82:18 which corresponds to aconcentration of about 12.5%, and up to 87.5/12.5 ratio, whichcorresponds to a concentration of about 9% (starting material/totalweight).

The X-ray diffraction analyses were performed using the conditions setout in Example 4. The results are shown in Table 16 below and thediffractograms obtained after the analysis of the solid phases insuspension at the end of the ethanol addition are presented in FIGS. 10ato 10c . These show diffraction profiles with no signal of Form Aobserved for the two tests with a final ethanol/water ration at 75:25and the test at 87.5:12.5. A small shoulder on the left of the peak at2θ=25.4° (corresponding to Form A) for the test at 82:18.

FIGS. 10a-c show XRPD diffractograms for, beginning at the lowest line:

Reference of Form A (bottom line in FIGS. 10a-c )

Reference of Form B

PE1716E007-L-6

PE1716E007-L-7

PE1716E007-L-8

PE1716E007-L-9 (top line in FIGS. 10a-c )

TABLE 16 Results of the crystallization tests by programmed addition m mInitial Final starting solvent Concentr. Add. Concentr. Test materialmixture (mg/mg Seeds Solvent added rate (mg/mg XRD Profile reference(mg) (mg) solution) (mg) (μL) (mg) (mL/min) solution) XRD referenceE007-L-6 750.24 1449.99 0.341 2.93 3000 2367.0 0.05 0.164 Form BPE1716X048 E007-L-7 749.20 1448.69 0.341 5.29 3000 2367.0 0.1 0.164 FormB PE1716X049 E007-L-8 750.57 1445.90 0.342 2.90 4844 3821.9 0.1 0.125Form B + Form A (weak) PE1716X050 E007-L-9 749.22 1445.08 0.341 2.987783 6140.8 0.1 0.090 Form B PE1716X051

Example 8: Crystallisation Process

An overview of the synthesis of triethylenetetramine (trientine)tetrahydrochloride (TETA 4HCl) is shown in the scheme below.

In Step I. triethylenetetramine (TETA) is converted to the correspondingtriethylenetetramine hydrate (TETA hydrate) by stirring in the presenceof water and TBME. Isopropanol is added as an anti-solvent and ifrequired, seeded with TETA hydrate. The TETA hydrate is crystallised,filtered and isolated. The crude triethylenetetramine tetrahydrochloride(Crude TETA 4HCl) is obtained by reaction of triethylenetetraminehydrate (TETA hydrate) with aqueous hydrochloric acid in ethanol (StepII). The crude triethylenetetramine tetrahydrochloride (Crude TETA 4HCl)is recrystallised from a mixture of purified water and ethanol. Thecrude triethylenetetramine tetrahydrochloride (Crude TETA 4HCl) isfurther purified by recrystallisation from a mixture of purified waterand ethanol in the presence of Form B seeds to give triethylenetetraminetetrahydrochloride (TETA 4HCl) (Step III).

The method produces a batch size of 110-130 kg TETA 4HCl, from 125 kg ofTETA. The overall yield for the synthesis is approximately 50% includingtwo recrystallisations of crude TETA 4HCl. The recrystallization toproduce Form B crystals is carried out as summarised in the flow chartsof FIGS. 1a, 1b and 2, but with the addition of a furtherrecrystallization of the crude TETA.4HCl at the end of Step II (FIG. 1b) and before Step III (FIG. 2). The process can be described as follows:

Once inertisation of the installations has been performed, allmanipulations are performed under nitrogen flow.

Step I: Manufacture of Triethylenetetramine Hydrate (TETA Hydrate)

-   -   Triethylenetetramine (nominal quantity 125 kg) is charged into a        reactor followed by TBME (185±5 kg). Water (ca 28 kg) is added        with stirring over ≥15 minutes whilst maintaining the        temperature at ≤30° C.    -   The solution is seeded with triethylenetetramine hydrate (ca.        0.1 kg) whilst stirring at 25-35° C., if required, to promote        crystallisation.    -   Isopropanol (64±1 kg) is added at 25-35° C.    -   The suspension is heated at 30-40° C. for ≥15 minutes, followed        by a slow cooling over ≥90 minutes to 15-25° C.    -   The suspension is cooled to −5 to 5° C. and is stirred for ≥30        minutes    -   The product is filtered and centrifuged. Then, a sample is taken        for analysis (GC Assay) and determination of impurities.    -   If the sample is sufficiently pure, the wet TETA hydrate is        filled in the dryer and dried at ≤25° C. until it meets the        requirements set out in the next step.    -   The dried product is analysed for purity by GC, appearance,        residual water by KF, identity by FTIR, sulphated ash and        residual solvents by GC.

Step II: Manufacture of Crude Triethylenetetramine Tetrahydrochloride(Crude TETA.4HCl)

-   -   Triethylenetetramine hydrate (TETA hydrate) is dissolved in        water (85±1 kg) and acidified with concentrated aqueous        hydrochloric acid (200±5 kg) charged over ≥1 h at ≤40° C. The pH        value is checked (target pH=1.0) and concentrated aqueous        hydrochloric acid is added until pH≤1.0 is met.    -   The reaction mixture is cooled to 15-25° C. and stirred for ≥10        minutes.    -   The solution is treated with ethanol (672±5 kg) which is charged        over ≥1.5 h, maintaining the temperature at ≤30° C.    -   The suspension is cooled to −5 to 5° C. and stirred for ≥30        minutes.    -   The product is filtered and the solid washed successively with        ethanol (1×20 kg, then 3×25 kg).    -   Crude triethylenetetramine tetrahydrochloride (crude TETA.4HC)        is dissolved in water (340±10 kg).    -   The solution is treated with ethanol (909±10 kg) which is        charged over ≥1.5 h, maintaining the temperature at ≤30° C.    -   The suspension is cooled to −5 to 5° C. and stirred for ≥30        minutes.    -   The product is filtered and the solid washed successively with        ethanol (1×14 kg, then 3×15 kg).

Step III: Manufacture of Triethylenetetramine Tetrahydrochloride(TETA.4HCl)

-   -   Crude triethylenetetramine tetrahydrochloride (crude TETA.4HC)        is dissolved in water (340±10 kg).    -   The solution is treated with ethanol (909±15 kg) which is        charged over ≥1.5 h, maintaining the temperature at 7-13° C.    -   The solution is seeded with TETA.4HCl (2 wt %) during the        ethanol addition.    -   The suspension is stirred for ≥5 hours, then cooled to −5° C.        and stirred for ≥30 minutes.    -   The product is filtered and the solid washed successively with        ethanol (1×14 kg, then 3×15 kg).    -   A sample of the product is analysed for purity by GC.    -   The product is dried at ≤40° C. and if the control parameter for        loss-on-drying is met, the product is milled.    -   The milled drug substance is transferred under nitrogen in        double food quality polyethylene bag and then placed in an        aluminium bag and sealed. The aluminium bag is inserted in a        HDPE drum.

Reprocessing

TETA 4HCl obtained after recrystallisation is tested for impurities byGC. If levels of impurities are too high, Step III can be repeated.

The invention claimed is:
 1. A crystalline form of triethylenetetraminetetrahydrochloride Form B having at least one of the followingcharacteristics: (i) an XRPD pattern having at least two peaks selectedfrom the peaks at 22.9, 25.4, 25.8, 26.6, 34.6 and 35.3±0.1°2θ; and (ii)a Raman spectrum having at least two peaks selected from the peaks at aRaman shift of 943, 1173, 1527 and 1612±5 cm⁻¹; wherein the crystallineform contains no more than 10 wt % of triethylenetetraminetetrahydrochloride Form A having an XRPD pattern having peaks at 25.2and 35.7±0.1°2θ; and XRPD pattern peaks are as measured using awavelength of 1.5418 Å.
 2. The crystalline form according to claim 1,having an XRPD pattern having peaks at 25.4, 34.6 and 35.3±0.1°2θ. 3.The crystalline form according to claim 1, which contains at least 95 wt% of triethylenetetramine tetrahydrochloride Form B having: (i) an XRPDpattern as defined in claim 1; and/or (ii) a Raman spectrum having atleast two peaks selected from the peaks at a Raman shift of 943, 1173,1527 and 1612±5 cm⁻¹.
 4. A pharmaceutical composition comprising thecrystalline form according to claim 1 and a pharmaceutically acceptablecarrier or diluent.
 5. The pharmaceutical composition according to claim4, which contains no more than 5 wt % triethylenetetraminetetrahydrochloride Form A having an XRPD pattern having peaks at 25.2and 35.7±0.1°2θ.
 6. The pharmaceutical composition according to claim 5,which contains no more than 2 wt % triethylenetetraminetetrahydrochloride Form A having an XRPD pattern having peaks at 25.2and 35.7±0.1°2θ.
 7. The pharmaceutical composition according to claim 5which contains no more than 1 wt % triethylenetetraminetetrahydrochloride Form A having an XRPD pattern having peaks at 25.2and 35.7±0.1°2θ.
 8. A solid oral dosage composition comprising thecrystalline form according to claim 1 and a pharmaceutically acceptablecarrier.
 9. A solid oral dosage composition comprising the crystallineform according to claim 2 and a pharmaceutically acceptable carrier. 10.A solid oral dosage composition comprising the crystalline formaccording to claim 3 and a pharmaceutically acceptable carrier.